SECTION ON MOLECULAR PATHOPHYSIOLOGY Medications development: Target discovery and validation. This group of projects utilizes rodent models, molecular studies and pharmacological approaches to identifying novel mechanisms and treatment targets, and validating them for human development. Two categories of models for target discovery Genetically selected alcohol preferring lines Prior behavioral characterization has shown that the AA line (collaboration with Dr. Hyyti, Helsinki, Finland) has a high degree of impulsivity, and dysfunctional endocannabinoid function in medial prefrontal cortex (mPFC), a region strongly implicated in impulse control. Recently, we found that in response to an alcohol challenge, AA rats show enhanced phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) in mPFC, with corresponding increased phosphorylation of AKT, a major regulator of GSK-3beta . In the non-preferring counterpart ANA line we found a weaker, AKT-independent phosphorylation of GSK-3beta by ethanol. AA rats showed a rapid and transient dephosphorylation of ERK1/2 in the medial prefrontal cortex (mPFC) and to a lesser degree in the nucleus accumbens;ANA rats were completely non-responsive for this mechanism. Together, these results emphasize the importance of the mPFC in the AA model (Neznanova et al, 2009) In contrast, the msP line is characterized by high stress reactivity / anxiety as well as high alcohol preference, making it a phenocopy of post-dependent animals (see below). Prior work has shown that this is related to an up-regulation of CRH1 receptor expression. Recent data point to nociceptin (NOP) participation in the cascade that drives the behavioral traits in this line (see below). Long-term neuroadaptations Prolonged exposure of the rat brain to repeated cycles of intoxication and mild withdrawal results in a long-term up-regulation of voluntary alcohol intake, encoded by long-term changes in gene expression patterns (Rimondini et al. 2002). Animals with a history of dependence have a long-lasting sensitization of stress responses;and respond to stress with increased voluntary alcohol consumption. A major driver for these behavioral changes is an up-regulation of the CRH system, primarily within the amygdala (reviewed in Heilig and Koob 2007). A persistent functional tolerance to alcohol is an important part of the post-dependent behavioral syndrome (Rimondini et al. 2008). Discovery and validation of novel targets: focus on stress and negative affect Stress is triggers relapse in alcoholics and in animal models. Several stress-related neuropeptide systems are candidate targets selectively sensitive in the post-dependent state. Corticotropin-Releasing Hormone (CRH) and its CRH1 receptor Both the elevated self-administration of alcohol and the increased behavioral sensitivity to stress in the post-dependent state is in large part mediated by an up-regulation of the CRH1 subtype of CRH receptors in the amygdala (Sommer et al. 2008). This converges with prior findings of a stress-sensitive / anxious behavioral phenotype in the msP rat, driven by an innate up-regulation of CRH1 receptors in the amygdala and several other brain regions in this line (Hansson et al. 2006). The elevated innate CRH1 expression of msP rats is rescued by voluntary alcohohol consumption (Hansson et al. 2007). Under a CRADA with Eli Lilly and Co, we have identified a series of CRH antagonists with suitable properties for clinical development (Gehlert et al. 2007;Thorsell et al, unpublished data).Under an agreement with BMS, we have evaluated another series of orally available, brain penetrant CRH1 antagonists to determine their suitability for use in forthcoming human trials. Neuropeptide Y (NPY) NPY is a potent endogenous anti-stress compound and counteracts the behavioral stress effects of CRH. Agonism on NPY-Y1 receptors, or blockade of presynaptic Y2 autoreceptors, also suppresses post-dependent drinking, while leaving basal intake of alcohol in non-dependent animals unaffected (reviewed in Thorsell et al. 2006). Under a CRADA with Johnson and Johnson Pharmaceuticals, we have evaluated of a non-peptide compound targeting the Y2 receptor to determine its potential for clinical development. Based on the results, the compound examined has been terminated. A collaboration with Scripps Florida under a UO1 mechanism is underway to develop novel compounds with improved properties. Substance P and its NK1 receptor The prototypical member of the neurokinin family, Substance P, and its preferred neurokinin-1 (NK1) receptor are expressed throughout the fear-processing pathways of the brain, Blockade of NK1 receptors suppresses behavioral stress responses in experimental animals. NK1R mutants showed markedly decreased alcohol preference, and this predicted human activity in a surrogate marker trial (George et al 2008). In follow-up work, we have found that conditioned place preference for alcohol, as well as escalation of intake over time are also eliminated, in a gene-dose dependent manner in the mutants (Thorsell et al. in press PNAS). Collaborating with Dr. Kenner Rice, we have resynthesized a literature compound that has been engineered to possess high affinity for the rat NK1 receptor. Using this compound, we have initiated rat pharmacology studies to better understand this mechanism. Initial data indicate a potent, dose dependent suppression of stress-induced reinstatement of alcohol seeking by the antagonist, an effect thought to be predictive of clinical anti-relapse activity. Nociceptin Nociceptin is the endogenous ligand for a GPCR, NOP, that belongs to the opioid recepto family but does not bind classical opioid peptides. NOP activation has both anti-opioid and anti-stress effects, making this receptor a candidate alcoholism treatment target. We collaborate around this target with the lab of Dr. Roberto Ciccocioppo. Recent work demonstrated that in the msP rat, a disruption of NOP signalling in central amygdala contributes to excessive alcohol intake, and can be rescued by local microinjections into this structure (Economidou et al. 2008). We are currently evaluating small molecules for their ability to reproduce the effects of nociceptin in alcohol paradigms. Support to a relevance of this target is provided by finding that a marker at the nociceptin gene locus is associated with alcohol dependence (Huang et al. 2008). Neuropeptide S (NPS) This is a novel exploratory project. The NPS receptor is a recently deorphanized GPCR that is unique in that its activation is at the same time anxiolytic and increased locomotor activity. Initial work in collaboration with the Ciccocioppo laboratory indicates that NPS signaling may have a role in relapse to alcohol seeking (Canella et al. 2009). We have developed a screenable assay for the NPS receptor, and in collaboration with Dr. Austin of the NIHGR Chemical Genomics Center completed a screen of their library. Leads are currently being optimized. Glutamatergic genes A hyperglutamatergic state evolves in alcohol dependence. GLAST clears extracellular glutamate, and is up-regulated in the post-dependent state. We have studied GLAST null-mutants, with the hypothesis that these might provide a model of the neuroadapted dependent state. Unexpectedly, we have found that GLAST null mutants, rather than displaying excessive alcohol intake, have markedly decreased intake levels, and decreased conditioned place preference for alcohol (Karlsson et al in preparation). These animals also display hyperlocomotion, which is rescued both by conventional antipsychotics as well as agonists at presynaptic mGluR2/3 autoreceptors that inhibit release of glutamate (Karlsson et al, 2008). Relevance These studies identify targets for alcoholism treatment